Transgenic cells with increased plastoquinone levels and methods of use

ABSTRACT

Disclosed herein are transgenic cells expressing a heterologous nucleic acid encoding a prephenate dehydrogenase (PDH) protein, a heterologous nucleic acid encoding a homogentisate solanesyl transferase (HST) protein, a heterologous nucleic acid encoding a deoxyxylulose phosphate synthase (DXS) protein, or a combination of two or more thereof. In particular examples, the disclosed transgenic cells have increased plastoquinone levels. Also disclosed are methods of increasing cell growth rates or production of biomass by cultivating transgenic cells expressing a heterologous nucleic acid encoding a PDH protein, a heterologous nucleic acid encoding an HST protein, a heterologous nucleic acid encoding a DXS protein, or a combination of two or more thereof under conditions sufficient to produce cell growth or biomass.

CROSS REFERENCE TO RELATED APPLICATIONS

This claims the benefit of U.S. Provisional Application No. 61/836,045,filed Jun. 17, 2013, which is incorporated herein by reference in itsentirety.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Contract No.DEAC52-06NA25396 awarded by the U.S. Department of Energy. Thegovernment has certain rights in the invention.

FIELD

This disclosure relates to transgenic cells and methods of use,particularly transgenic cells having increased levels of plastoquinone.

BACKGROUND

Environmentally sustainable energy production and reduction ofgreenhouse gas emissions can be achieved by use of carbon-neutral energysources. Wind, geothermal, solar, hydroelectric, and biofuels are nowbeing developed as sustainable sources of domestic energy production.Unlike wind, solar, or hydroelectric energy sources, biofuels can beconverted into energy-dense, liquid fuels that are compatible withcurrent energy distribution and consumption systems. Since biofuels havethe potential to reduce global carbon emission, they are an attractivepart of the mix of sustainable energy solutions. In addition, oil-basedbiofuels are one of the few renewable, energy-dense fuels that can beused by the aviation and shipping transportation sectors. However,demands for food and environmental sustainability limit the use of cropsor plants for biofuel production.

Unicellular algae are a prime candidate for production of biofuels. Incontrast to plants, unicellular algae do not partition large amounts ofbiomass into supportive structures such as stems and roots. Under nearideal growth conditions algae direct most of their energy into celldivision (6-12 hour cycle), allowing for rapid biomass accumulation.Under stress conditions (e.g., low nitrogen) or in the presence ofexogenous reductants (sugar, glycerol) metabolism is redirected towardsthe production of energy-dense storage compounds such as lipids. Manyunicellular algae are facultatively capable of producing 4% to 60%lipids per gram dry weight under the appropriate growth conditions(e.g., stress or photoheterotrophic growth), making them one of the mostefficient biofuel production systems known. It has been estimated that,on area basis, algae may produce up to twenty times the fuel of any landplant system. Despite these useful characteristics, many challengesremain for developing efficient, large-scale production of biofuel fromalgae.

SUMMARY

Disclosed herein are transgenic photosynthetic cells (for example plantor algae cells) and methods of making and using such cells. In someexamples, the transgenic cells express a heterologous nucleic acidencoding a prephenate dehydrogenase (PDH) protein, a heterologousnucleic acid encoding a homogentisate solanesyl transferase (HST)protein, a heterologous nucleic acid encoding a deoxyxylulose phosphatesynthase (DXS) protein, or a combination of two or more thereof. In someexamples, the transgenic cells express a heterologous nucleic acidencoding a PDH protein and a heterologous nucleic acid encoding a HSTprotein. In some embodiments, the transgenic photosynthetic cells areplant cells (such as canola or Camelina cells). In other embodiments,the transgenic photosynthetic cells are algae cells (such asChlamydomonas cells).

The disclosed transgenic cells are useful for a variety of applications,including production of biofuels. In particular examples, the disclosedtransgenic cells have increased plastoquinone (PQ) levels compared to acontrol, for example compared to cells lacking the transgene(s)described herein. Increasing the amount of PQ in a photosynthetic cellincreases photosynthetic efficiency (for example by reducingnon-photochemical quenching (NPQ) by the xanthophylls cycle but increaseNPQ through direct quenching of chlorophyll excited states, and/orincreasing photochemical quenching), increases cell growth rates, and/orincreases production of biomass. Therefore, disclosed herein are methodsof increasing PQ levels in photosynthetic cells (such as plant or algaecells) by expressing a heterologous nucleic acid encoding a PDH protein,a heterologous nucleic acid encoding an HST protein, a heterologousnucleic acid encoding a DXS protein, or a combination of two or morethereof (such as a heterologous nucleic acid encoding a PDH protein anda heterologous nucleic acid encoding an HST protein).

The disclosed methods also include methods of increasing cellular growthrates or production of biomass compared to a control (for examplecompared to cells lacking the transgene(s) described herein) bycultivating a transgenic cell expressing a heterologous nucleic acidencoding a PDH protein, a heterologous nucleic acid encoding an HSTprotein, a heterologous nucleic acid encoding a DXS protein, or acombination of two or more thereof (such as a heterologous nucleic acidencoding a PDH protein and a heterologous nucleic acid encoding an HSTprotein) under conditions sufficient to produce cell growth or biomass.

The foregoing and other features of the disclosure will become moreapparent from the following detailed description, which proceeds withreference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing a portion of the biosyntheticpathway for plastoquinone in algae. PDH, prephenate dehydrogenase; HPPD,4-hydroxyphenyl pyruvate dioxygenase; HST, homogentisate solanesyltransferase; DXS, deoxyxylulose phosphate synthase.

FIGS. 2A and 2B are a pair of diagrams of PDH and HST expressionvectors, respectively, used to generate transgenic Chlamydomonasreinhardtii cell lines.

FIGS. 3A and 3B are a pair of digital images of gels showing PCRidentification of positive transgenic PDH or HST lines aftertransformation and selection on HS media (FIG. 3A) or RT-PCRconfirmation of selected PDH transgenic cell lines (FIG. 3B). Checkmarks indicate positive transgenic lines; “X” marks indicate negativecell lines, “+” indicates plasmid positive control, and “−” indicatesnegative control.

FIG. 4 is a graph showing chlorophyll fluorescence kinetics ofWT-complement (WT-c), HST transgenic, and PDH transgenic algae linesbefore and after an actinic flash.

FIGS. 5A-5C are a series of graphs showing quinone reoxidation kineticsof WT-c (FIG. 5A), PDH1-4 line (FIG. 5B), and PDH2-16 line (FIG. 5C).The curves were fit using three independent exponential rate constantsusing the equations shown. A=amplitude of rate constant. T=rate constantin seconds.

FIGS. 6A and 6B are a pair of graphs showing fluctuating (FIG. 6A) andregular (FIG. 6B) light-dark regimes for algae growth.

FIG. 7 is a graph showing growth of three PDH transgenic and two WT-ccell lines under fluctuating light conditions in Phenometrics (15 cmdeep) photobioreactors.

FIG. 8 is a schematic diagram of a PDH/HST expression vector used togenerate transgenic Chlamydomonas reinhardtii cell lines.

FIG. 9 is a digital image of a gel showing PCR identification ofpositive transgenic PDH/HST lines after transformation and selection onHS media. Check marks indicate positive transgenic lines; “+” indicatesplasmid positive control.

FIG. 10 is a graph (top) and table (bottom) showing raw chlorophyllfluorescence induction kinetics of two PDH/HST transgenic cell lines(PH-6 and PH-14) and a wild-type cell line (WTc-38).

FIG. 11 is a graph showing chlorophyll fluorescence induction kineticsin two PDH/HST transgenic cell lines (PH-6 and PH-14) and a wild-typecell line (WTc-38) in the presence and absence of the photosystem IIinhibitor atrazine (10 μM).

SEQUENCE LISTING

Any nucleic acid and amino acid sequences listed herein or in theaccompanying sequence listing are shown using standard letterabbreviations for nucleotide bases and amino acids, as defined in 37C.F.R. §1.822. In at least some cases, only one strand of each nucleicacid sequence is shown, but the complementary strand is understood asincluded by any reference to the displayed strand.

The Sequence Listing is submitted as an ASCII text file in the form ofthe file named Sequence_Listing.txt, which was created on Jun. 16, 2014,and is 11,577 bytes, which is incorporated by reference herein.

SEQ ID NOs: 1 and 2 are an exemplary codon optimized PDH nucleic acidsequence and a PDH amino acid sequence, respectively.

SEQ ID NOs: 3 and 4 are exemplary HST nucleic acid and amino acidsequences, respectively.

SEQ ID NOs: 5 and 6 are nucleic acid sequences of forward and reverseprimers, respectively, for amplification of PDH or HST in transgenicalgae.

SEQ ID NOs: 7 and 8 are nucleic acid sequences of forward and reversepsbA primers, respectively.

SEQ ID NOs: 9 and 10 are nucleic acid sequences of forward and reversePDH primers, respectively

DETAILED DESCRIPTION

The slowest step (1 ms) in photosynthetic electron transfer is theoxidation of dihydroplastoquinone (plastoquinol; PQH₂) by the cytochromeb6f (Cytb6f) complex. PQ plays a critical role in buffering the fastrate (1 μs) of photosystem II (PSII) electron transfer and the slow rate(1 ms) of PQH₂ oxidation by the Cytb₆f complex. The PQ pool size inthylakoid membranes is small, about 8 PQ/PSII/Cytb6f complex (Kruk andKarpinski, Biochim. Biophys. Acta 1757:1669-1675, 2006). PQ also has alimited half-life in the membrane of about 9 hours (Wanke et al., PlantSci. 154:183-187, 2000). It is shown herein that by increasing thethylakoid PQ pool size (e.g., in algae cells), the electron and protonbuffering capacity of the thylakoid membrane and threshold lightintensity at which non-photochemical quenching is activated areincreased. Without being bound by theory, it is believed that increasedPQ pool size enhances energy conversion efficiency by indirectlyreducing the optical cross section of the light harvesting antennaecomplex by quenching excess chlorophyll excited states at high lightintensities, reducing generation of reactive oxygen species which damagethe photosynthetic machinery. In addition, the larger PQ pool sizebuffers transient fluxes in electron transfer rates in non-equilibriumsituations, such as fluctuating light intensities.

The rate of PQH₂ oxidation by the Cytb6f complex is controlled in partby the rate of PQH₂ diffusion between PSII and the Cytb6f complex. Ithas been estimated by percolation theory that the rate of PQH₂ diffusionin thylakoid membranes is 1000 times slower than in liposomes due thepresence of dispersed macromolecular protein complexes (Kirchhoff etal., Biochemistry 41:4872-4882, 2002). By creating thylakoid membranemicro-domains with elevated PQH₂ concentrations or by channeling PQH₂transfer, the rate of PQH₂ reduction and oxidation is enhanced.

The synthetic pathway for PQ in algae is shown in FIG. 1. The pathwayconverts prephenate to arogenate, then tyrosine, and ultimately4-hydroxyphenylpyruvate. The pathway then branches for production oftocopherols or PQ. For production of PQ, 4-hydroxyphenylpyruvate isconverted to homogentisate by 4-hydroxyphenylpyruvate dioxygenase.Homogentisate is converted to 2-demethylplastoquinol-9 by HST and thenby reduction and oxidation to plastoquinol-9 (PQ). In some embodimentsdisclosed herein, the conversion of prephenate to4-hydroxyphenylpyruvate, which normally involves several steps in algae,is achieved in a single step by expression in the algae of aheterologous PDH, for example from yeast. This drives the pathway to thetocopherol/PQ branchpoint, increasing PQ production. In addition, theexpression of PDH in chloroplasts may alter the synthesis of aromaticamino acids and their pool sizes. Increasing expression of HST in thealgae (for example, by expressing a heterologous HST nucleic acid oroverexpressing native algae HST) either separately or simultaneouslywith PDH expression can also increase PQ production by driving thetocopherol/PQ branchpoint toward the PQ pathway. PQ production in thealgae can also be increased by increasing expression of DXS, which isinvolved in synthesis of isopentenyl diphosphate, the precursor forisoprenoids, including plastoquinone.

I. Abbreviations

Chl chlorophyll

Cytb6f cytochrome b6f complex

DXS deoxyxylulose phosphate synthase

HS high salt medium

HST homogentisate solanesyl transferase

NPQ non-photochemical quenching

PDH prephenate dehydrogenase

PQH₂ dihydroplastoquinone (plastoquinol)

PQ plastoquinone

PSII photosystem II

II. Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. It is further tobe understood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of this disclosure, suitable methods andmaterials are described below. The term “comprises” means “includes.”Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Allsequence database accession numbers (such as GenBank, EMBL, or UniProt)mentioned herein are incorporated by reference in their entirety aspresent in the respective database on Jun. 17, 2013. In case ofconflict, the present specification, including explanations of terms,will control. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

In order to facilitate review of the various embodiments of theinvention, the following explanations of specific terms are provided:

Algae: A group of autotrophic organisms which range from unicellular tomulticellular forms. Unicellular algae are commonly referred to asmicroalgae. Microalgae include Achnanthes, Amphora, Borodinella,Botryococcus, Chaetoceros, Chlorococcum, Chlorella, Chlamydomonas,Cyclotella, Dunaliella, Galdieria, Pleurochrysis, Monoraphidium,Nannochloris, Nannochloropsis, Navicula, Nitzschia, Oocysitis,Oscillatoria, Phaeodactylum, Scenedesmus, Stichococcus, Synechococcus,Tetraselmis, and Thalassiosira. Macroalgae (seaweed) include Gracilariaand Sargassum.

Biomass: Biological material from living or recently living organisms.In some examples, biomass is the mass of living biological organisms ina given area, volume, or ecosystem at a given time. Biomass is theamount of cellular material in a cultivation system (such as abioreactor, pond, or other container) at a point in time. The amount ofbiomass can be determined by the number of cells present, wet weight,dry weight, amount of a cellular constituent (such as chlorophyll-a),absorbance (e.g., OD₇₅₀), or any other measurement known to one ofordinary skill in the art.

Conservative variants: A substitution of an amino acid residue foranother amino acid residue having similar biochemical properties.“Conservative” amino acid substitutions are those substitutions that donot substantially affect or decrease an activity of a polypeptide (suchas PDH polypeptide, an HST polypeptide, or a DXS polypeptide). Apolypeptide can include one or more amino acid substitutions, forexample 1-10 conservative substitutions, 2-5 conservative substitutions,4-9 conservative substitutions, such as 1, 2, 5 or 10 conservativesubstitutions. Specific, non-limiting examples of a conservativesubstitution include the following examples (Table 1).

TABLE 1 Exemplary conservative amino acid substitutions Very Highly -Original Conserved Highly Conserved Substitutions ConservedSubstitutions Residue Substitutions (from the Blosum90 Matrix) (from theBlosum65 Matrix) Ala Ser Gly, Ser, Thr Cys, Gly, Ser, Thr, Val Arg LysGln, His, Lys Asn, Gln, Glu, His, Lys Asn Gln; His Asp, Gln, His, Lys,Ser, Thr Arg, Asp, Gln, Glu, His, Lys, Ser, Thr Asp Glu Asn, Glu Asn,Gln, Glu, Ser Cys Ser None Ala Gln Asn Arg, Asn, Glu, His, Lys, Met Arg,Asn, Asp, Glu, His, Lys, Met, Ser Glu Asp Asp, Gln, Lys Arg, Asn, Asp,Gln, His, Lys, Ser Gly Pro Ala Ala, Ser His Asn; Gln Arg, Asn, Gln, TyrArg, Asn, Gln, Glu, Tyr Ile Leu; Val Leu, Met, Val Leu, Met, Phe, ValLeu Ile; Val Ile, Met, Phe, Val Ile, Met, Phe, Val Lys Arg; Gln; GluArg, Asn, Gln, Glu Arg, Asn, Gln, Glu, Ser, Met Leu; Ile Gln, Ile, Leu,Val Gln, Ile, Leu, Phe, Val Phe Met; Leu; Tyr Leu, Trp, Tyr Ile, Leu,Met, Trp, Tyr Ser Thr Ala, Asn, Thr Ala, Asn, Asp, Gln, Glu, Gly, Lys,Thr Thr Ser Ala, Asn, Ser Ala, Asn, Ser, Val Trp Tyr Phe, Tyr Phe, TyrTyr Trp; Phe His, Phe, Trp His, Phe, Trp Val Ile; Leu Ile, Leu, Met Ala,Ile, Leu, Met, Thr

The term conservative variation also includes the use of a substitutedamino acid in place of an unsubstituted parent amino acid, provided thatthe substituted polypeptide retains an activity of the unsubstitutedpolypeptide.

Control: A sample or standard used for comparison with an experimentalsample. In some examples, the control is a cell (or a culture of cells)that does not contain one or more transgenes which are included in thecell (or culture) for comparison. For example, a cell that does notcontain a heterologous PDH nucleic acid can be a control for a cell thatcontains a heterologous PDH nucleic acid. In some examples, the controlcell is a wild type (or non-transgenic) cell. In other examples, thecontrol cell is a transgenic cell, but one that does not contain atleast one transgene of interest. For example, a cell that contains aheterologous PDH nucleic acid can be a control for a cell that containsa heterologous HST nucleic acid, but does not contain a heterologous PDHnucleic acid. In still other examples, a control cell may be a wild typecomplement cell, such as a deletion mutant (such as a psbA deletionmutant) that is transformed with a nucleic acid encoding the deletednucleic acid, but not any additional nucleic acid, such as aheterologous PDH, HST, DXS, or other nucleic acid.

Cultivation or Culturing: Intentional growth of an organism or cell,such as an alga (for example, Chlamydomonas) or plant cell in thepresence of assimilable sources of carbon, nitrogen, and mineral salts.In an example, such growth can take place in a solid or semi-solidnutritive medium, or in a liquid medium in which the nutrients aredissolved or suspended. In a further example, the cultivation may takeplace on a surface or by submerged culture. The nutritive medium can becomposed of complex nutrients or can be chemically defined.

Deoxyxylulose phosphate synthase (DXS): An enzyme that catalyzes thethiamin diphosphate-dependent condensation of pyruvate andD-glyceraldehyde-3-phosphate to yield 1-deoxy-D-xylulose 5-phosphate.1-deoxy-D-xylulose 5-phosphate is converted to isopentenyl diphosphate,which is the precursor for isoprenoids, including plastoquinone. DXSnucleic acid and amino acid sequences are publicly available. One ofordinary skill in the art can identify DXS nucleic acid and amino acidsequences. Exemplary, non-limiting, DXS sequences are provided herein.

Expression: Transcription or translation of a nucleic acid sequence. Forexample, a gene is expressed when its DNA is transcribed into an RNA orRNA fragment, which in some examples is processed to become mRNA. A genemay also be expressed when its RNA or mRNA is translated into an aminoacid sequence, such as a protein or a protein fragment. In a particularexample, a heterologous gene is expressed when it is transcribed into anRNA. In another example, a heterologous gene is expressed when its RNAis translated into an amino acid sequence. The term “expression” is usedherein to denote either transcription or translation. Regulation ofexpression can include controls on transcription, translation, RNAtransport and processing, degradation of intermediary molecules such asmRNA, or through activation, inactivation, compartmentalization ordegradation of specific protein molecules after they are produced.

Gene: A segment of nucleic acid that encodes an individual protein orRNA molecule (also referred to as a “coding sequence” or “codingregion”) and may include non-coding regions (“introns”) and/orassociated regulatory regions such as promoters, operators, terminatorsand the like, that may be located upstream or downstream of the codingsequence.

Heterologous: Originating from a different genetic source or species orpresent at a genetic locus other than the naturally occurring geneticlocus in the organism. In some examples, a gene or nucleic acid that isheterologous to a cell originates from an organism or species other thanthe cell in which it is expressed (for example from a differentspecies). In other examples, a gene or nucleic acid that is heterologousto a cell is present at a different genetic locus (such as on adifferent chromosome, at a different location in a chromosome, orexogenous to a chromosome, such as on a plasmid) than the naturallyoccurring genetic locus in the cell. In further examples, a heterologousnucleic acid may include a duplication of a naturally occurring nucleicacid, such that two (or more) copies of the nucleic acid are present inthe cell or organism, for example at the genetic locus of the naturallyoccurring copy of the nucleic acid. Methods for introducing aheterologous gene or nucleic acid in a cell or organism are well knownin the art, for example transformation with a nucleic acid, includingelectroporation, lipofection, and particle gun acceleration.

Homogentisate solanesyl transferase (HST): Also known as homogentisateprenyltransferase. An enzyme capable of catalyzing the condensation ofhomogentisate and solanesyl diphosphate to form 2-demethylplastoquinol-9(e.g., Tian et al., Planta 226:1067-1073, 2007). In subsequent steps inthe PQ biosynthetic pathway, 2-demethylplastoquinol-9 is methylated andoxidized to form PQ. HST nucleic acid and amino acid sequences arepublicly available and can be identified by one of ordinary skill in theart. Exemplary, non-limiting, HST sequences are provided herein.

Isolated: An “isolated” biological component (such as a nucleic acidmolecule, protein, or cell) has been substantially separated or purifiedaway from other biological components, such as components in the cell ofthe organism, or the organism itself, in which the component naturallyoccurs, such as other chromosomal and extra-chromosomal DNA and RNA,proteins and cells. Nucleic acid molecules and proteins that have been“isolated” include nucleic acid molecules and proteins purified bystandard purification methods. The term also embraces nucleic acidmolecules and proteins prepared by recombinant expression in a host cellas well as chemically synthesized nucleic acid molecules and proteins.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein-coding regions, in the samereading frame. In some examples, a promoter sequence is operably linkedto a protein-encoding sequence, such that the promoter drivestranscription of the linked nucleic acid and/or expression of theprotein.

Photosynthetic cell: A cell that is able to convert light energy (forexample, solar energy) to chemical energy. Photosynthetic cells containlight sensitive pigments, such as chlorophyll, that capture solarenergy. Chlorophyll and other light sensitive pigments are typicallypresent in cells in chloroplast organelles. Exemplary photosyntheticcells are plant cells and algae cells (including macroalgae andmicroalgae cells).

Plant cell: Any cell derived from a plant, including cells fromundifferentiated tissue (e.g., callus) as well as plant seeds, pollen,propagules and embryos. Plant cells can be obtained from any plant organor tissue and cultures prepared therefrom.

Plastoquinone (PQ): A quinone molecule involved in the electrontransport chain of photosynthesis. Plastoquinone is reduced (accepts twoprotons (H⁺) from the stromal matrix of the chloroplast, coupled to twoelectrons (e⁻) from photosystem II), forming plastoquinol. It transportsthe protons to the lumen of thylakoid discs, while the electronscontinue through the electron transport chain into the cytochrome b6fprotein complex. Plastoquinones have from six to nine isoprenoid units.Plastoquinone-9 has the structure:

Prephenate dehydrogenase (PDH): Also known as Tyr1. An enzyme capable ofcatalyzing the transformation of prephenate to 4-hydroxyphenylpyruvate.4-hydroxyphenylpyruvate is converted to homogentisate, by hydroxyphenyldioxygenase. Homogentisate is the branchpoint for synthesis oftocopherols and PQ. PDH nucleic acid and amino acid sequences arepublicly available and can be identified by one of ordinary skill in theart. Exemplary, non-limiting, PDH sequences are provided herein.

Promoter: Promoters are sequences of DNA near the 5′ end of a gene thatact as a binding site for RNA polymerase, and from which transcriptionis initiated. A promoter includes necessary nucleic acid sequences nearthe start site of transcription, such as, in the case of a polymerase IItype promoter, a TATA element. In one embodiment, a promoter includes anenhancer. In another embodiment, a promoter includes a repressorelement.

Promoters may be constitutively active, such as a promoter that iscontinuously active and is not subject to regulation by external signalsor molecules. In some examples, a constitutive promoter is active suchthat expression of a sequence operably linked to the promoter isexpressed ubiquitously (for example, in all cells of a tissue or in allcells of an organism and/or at all times in a single cell or organism,without regard to temporal or developmental stage).

Promoters may be inducible or repressible, such that expression of asequence operably linked to the promoter can be expressed under selectedconditions. In some examples, a promoter is an inducible promoter, suchthat expression of a sequence operably linked to the promoter isactivated or increased. An inducible promoter may be activated bypresence or absence of a particular molecule, for example, tetracycline,metal ions, alcohol, or steroid compounds. An inducible promoter alsoincludes a promoter that is activated by environmental conditions, forexample, light or temperature. In further examples, the promoter is arepressible promoter such that expression of a sequence operably linkedto the promoter can be reduced to low or undetectable levels, oreliminated. A repressible promoter may be repressed by direct binding ofa repressor molecule (such as binding of the trp repressor to the trpoperator in the presence of tryptophan). In a particular example, arepressible promoter is a tetracycline repressible promoter. In otherexamples, a repressible promoter is a promoter that is repressible byenvironmental conditions, such as hypoxia or exposure to metal ions.

Purified: The term purified does not require absolute purity; rather, itis intended as a relative term. Thus, for example, a purifiedpreparation of a compound or a cell is one in which the specifiedcompound or cell is more enriched than it is in its generativeenvironment, for instance in a prokaryotic cell or in a cell culture(for example, in cell culture medium). Preferably, a preparation of aspecified compound is purified such that the compound represents atleast 50% of the total content of the preparation. In some embodiments,a purified preparation contains at least 60%, at least 70%, at least80%, at least 85%, at least 90%, at least 95% or more of the specifiedcompound.

Sequence identity: The similarity between two nucleic acid sequences, ortwo amino acid sequences, is expressed in terms of the similaritybetween the sequences, otherwise referred to as sequence identity.Sequence identity is frequently measured in terms of percentage identity(or similarity or homology); the higher the percentage, the more similarthe two sequences are.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smithand Waterman (Adv. Appl. Math., 2:482, 1981); Needleman and Wunsch (J.Mol. Biol., 48:443, 1970); Pearson and Lipman (Proc. Natl. Acad. Sci.,85:2444, 1988); Higgins and Sharp (Gene, 73:237-44, 1988); Higgins andSharp (CABIOS, 5:151-53, 1989); Corpet et al. (Nuc. Acids Res.,16:10881-90, 1988); Huang et al. (Comp. Appls. Biosci., 8:155-65, 1992);and Pearson et al. (Meth. Mol. Biol., 24:307-31, 1994). Altschul et al.(Nature Genet., 6:119-29, 1994) presents a detailed consideration ofsequence alignment methods and homology calculations.

The alignment tools ALIGN (Myers and Miller, CABIOS 4:11-17, 1989) orLFASTA (Pearson and Lipman, Proc. Natl. Acad. Sci. 85:2444-2448, 1988)may be used to perform sequence comparisons. ALIGN compares entiresequences against one another, while LFASTA compares regions of localsimilarity. These alignment tools and their respective tutorials areavailable on the Internet. Alternatively, for comparisons of amino acidsequences of greater than about 30 amino acids, the “Blast 2 sequences”function can be employed using the default BLOSUM62 matrix set todefault parameters, (gap existence cost of 11, and a per residue gapcost of 1). When aligning short peptides (fewer than around 30 aminoacids), the alignment should be performed using the “Blast 2 sequences”function, employing the PAM30 matrix set to default parameters (open gap9, extension gap 1 penalties). The BLAST sequence comparison system isavailable, for instance, from the NCBI web site; see also Altschul etal., J. Mol. Biol., 215:403-10, 1990; Gish and States, Nature Genet.,3:266-72, 1993; Madden et al., Meth. Enzymol., 266:131-41, 1996;Altschul et al., Nucleic Acids Res., 25:3389-402, 1997; and Zhang andMadden, Genome Res., 7:649-56, 1997.

Orthologs (equivalent to proteins of other species) of proteins are insome instances characterized by possession of greater than 75% sequenceidentity counted over the full-length alignment with the amino acidsequence of a specific protein using ALIGN set to default parameters.Proteins with even greater similarity to a reference sequence will showincreasing percentage identities when assessed by this method, such asat least 80%, at least 85%, at least 90%, at least 92%, at least 95%, atleast 98%, or at least 99% sequence identity.

When significantly less than the entire sequence is being compared forsequence identity, homologous sequences will typically possess at least80% sequence identity over short windows of 10-20, and may possesssequence identities of at least 85%, at least 90%, at least 95%, 96%,97%, 98%, or at least 99%, depending on their similarity to thereference sequence. Sequence identity over such short windows can bedetermined using LFASTA. One of skill in the art will appreciate thatthese sequence identity ranges are provided for guidance only; it isentirely possible that strongly significant homologs could be obtainedthat fall outside of the ranges provided. Similar homology conceptsapply for nucleic acids as are described for protein. An alternativeindication that two nucleic acid molecules are closely related is thatthe two molecules hybridize to each other under stringent conditions.

Nucleic acid sequences that do not show a high degree of identity maynevertheless encode similar amino acid sequences, due to the degeneracyof the genetic code. It is understood that changes in nucleic acidsequence can be made using this degeneracy to produce multiple nucleicacid sequences that each encode substantially the same protein.

Transduced and Transformed: A virus or vector “transduces” a cell whenit transfers nucleic acid into the cell. A cell is “transformed” by anucleic acid transduced into the cell when the DNA becomes stablyreplicated by the cell, either by incorporation of the nucleic acid intothe cellular genome, or by episomal replication. As used herein, theterm transformation encompasses all techniques by which a nucleic acidmolecule is introduced into such a cell, including transformation withplasmid vectors, and introduction of naked DNA by electroporation,lipofection, and particle gun acceleration.

Vector: A nucleic acid molecule as introduced into a host cell (such asan alga or plant cell), thereby producing a transformed host cell. Avector may include nucleic acid sequences that permit it to replicate ina host cell, such as an origin of replication. A vector may also includeone or more selectable marker genes and/or other genetic elements knownin the art. Vectors include plasmid vectors, including plasmids forexpression in plant or algae cells.

III. Transgenic Photosynthetic Cells

Disclosed herein are transgenic photosynthetic cells (for example, plantor algae cells). In various embodiments, the transgenic cells express aheterologous nucleic acid encoding a PDH protein, a heterologous nucleicacid encoding an HST protein, a heterologous nucleic acid encoding a DXSprotein, or a combination of two or more thereof. In some embodiments,the transgenic cells express a heterologous nucleic acid encoding a PDHprotein. In other embodiments, the transgenic cells express aheterologous nucleic acid encoding an HST protein. In additionalembodiments, the transgenic cells express a heterologous nucleic acidencoding a DXS protein. In still further embodiments, the transgeniccells express a heterologous nucleic acid encoding a PDH protein and aheterologous nucleic acid encoding an HST protein. In additionalembodiments, the transgenic cells express a heterologous nucleic acidencoding a PDH protein and a heterologous nucleic acid encoding a DXSprotein, the transgenic algae express a heterologous nucleic acidencoding an HST protein and a heterologous nucleic acid encoding a DXSprotein, or the transgenic algae express a heterologous nucleic acidencoding a PDH protein, a heterologous nucleic acid encoding an HSTprotein, and a heterologous nucleic acid encoding a DXS protein.

In some embodiments, the transgenic cells have increased PQ amounts, forexample as compared to control cell (such as a wild type(non-transgenic) cell of the same strain or species as the transgeniccell, or a transgenic cell that does not express the heterologous PDH,HST, and/or DXS protein). In some examples, the transgenic cellsdisclosed herein have at least about 5% increased PQ levels (such as atleast about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%,200%, 300%, 400%, 500%, or more) as compared to control cells. Forexample the transgenic cells may have about 5-500% more PQ (such asabout 10-400%, 20-300%, 50-250%, 75-200%, 50-150%, 10-100%, 10-75%, or10-50% more) than a control cell. Methods of determining the amount(quantitatively or relative to a control) of PQ in cells (such as aplant or alga cell or a population of plant or algae cells) are known toone of ordinary skill in the art. These methods include directmeasurements (for example, high pressure liquid chromatography or massspectrometry) or indirect measurements (such as chlorophyll fluorescenceor NPQ). In particular examples, the methods described in Example 2,below are used to determine an amount of PQ (such as an increase ordecrease in PQ compared to a control) in cells.

In some embodiments, transgenic cells with varying levels of PQ aregenerated to optimize improvements in photosynthetic energy conversionwhile maintaining synthesis of other necessary metabolites, such astocopherols and aromatic amino acids. In some examples, transgenic cellswith varying levels of PQ are produced by transforming cells with one ormore of the heterologous nucleic acids described herein and screeningthe resulting cell lines for PQ levels (directly or indirectly). Inother examples, transgenic cells with varying levels of PQ are producedby transforming cells with one or more of the heterologous nucleic acidsdisclosed herein under the control of promoters of varying strength (forexample, strong, medium, or weak promoters). Exemplary promoters for usein the transgenic cell lines are discussed below. The resulting celllines are screened for PQ levels (directly or indirectly), and celllines with the desired properties are selected. In some non-limitingembodiments, the selected cell lines have an increase of about 1-5-foldin PQ levels as compared to a control cell line.

In additional examples, the properties of the transgenic cells withrespect to PQ levels can be evaluated by oxygen evolution measurements.These studies are carried out on thylakoids, with varying time intervalsbetween flashes of light to induce charge separation and oxygenevolution. Without being bound by theory, it is believed that transgeniclines with an increased (e.g., a larger) PQ pool size will be able torestore the oxygen evolution capacity at shorter time intervals comparedto ones with a smaller PQ pool size (e.g., a control cell, or atransgenic cell with a smaller increase in the PQ pool size).

In other examples, the relative sensitivity of the transgenic cells tophotoinhibition, oxygen evolved is measured while exposing thethylakoids to high light intensities (for example, about 1000 μmoles for0-30 minutes). It is believed that transgenic cells with a larger PQpool size will produce more oxygen/[Chl] compared to those with asmaller PQ pool size (e.g., a control cell, or a transgenic cell with asmaller increase in the PQ pool size), and thereby be less sensitive tophotoinhibition (e.g., Ruffle et al., Plant Physiology 127:633-644,2001).

A. Prephenate Dehydrogenase (PDH)

Specific transgenic cells disclosed herein express a heterologous pdhnucleic acid. The pdh nucleic acid (such as all or a portion of a pdhgene) encodes a protein capable of catalyzing the transformation ofprephenate to 4-hydroxyphenylpyruvate. In non-limiting examples, the pdhnucleic acid or protein is a Saccharomyces cerevisiae pdh gene orprotein. Nucleic acid and amino acid sequences for pdh are publiclyavailable. For example, GenBank Accession Nos. NM_001178514, Z36035,DQ332878, and FN393060 (nucleotide 542696-544054 (reverse complement))disclose exemplary yeast pdh nucleic acid sequences. GenBank AccessionNos. NP_009725, CAA85127, and CBK39241 disclose exemplary yeast PDHamino acid sequences. In other examples, the PDH nucleic acid or proteinis from yeast (such as Saccharomyces, Schizosaccharomyces, Candida, orKluveromyces), a plant (such as Arabidopsis thaliana, Glycine max, Oryzasativa, or Zea mays), or bacterium (such as E. coli). Exemplary pdhnucleic acid sequences include GenBank Accession Nos. NM_001023517(Schizosaccharomyces), XM_717584 (Candida), XM_455066 (Kluveromyces),NM_101439 (Arabidopsis thaliana), XM_003545166 or XR_417861 (Glycinemax), NM_001065074 (Oryza sativa), and NM_001153997 (Zea mays), each ofwhich is incorporated herein by reference. One of ordinary skill in theart can identify additional PDH nucleic acid and amino acid sequences,for example, PDH sequences from other organisms.

In one non-limiting example, a pdh nucleic is from S. cerevisiae. Insome examples, the pdh nucleic acid includes or consists of the nucleicacid sequence set forth as:

(SEQ ID NO: 1) ATGGTATCAGAGGATAAGATTGAGCAATGGAAAGCCACAAAAGTCATTGGTATAATTGGTCTGGGTGATATGGGCCTATTATACGCTAATAAATTTACAGATGCTGGATGGGGTGTTATATGTTGTGATAGGGAAGAATATTATGATGAACTGAAAGAAAAATATGCCTCAGCTAAATTCGAACTGGTGAAAAATGGTCATTTGGTATCCAGGCAAAGCGACTATATTATCTATAGTGTTGAAGCATCCAATATTAGTAAGATCGTCGCAACGTATGGACCATCTTCTAAGGTTGGAACAATTGTTGGGGGTCAAACGAGTTGTAAGCTGCCGGAAATCGAGGCTTTCGAAAAGTATTTACCCAAGGACTGCGACATCATTACCGTGCATTCCCTTCATGGGCCTAAAGTTAATACTGAAGGCCAACCACTAGTTATTATCAATCACAGATCACAGTACCCAGAATCTTTTGAGTTCGTTAATTCTGTTATGGCATGTTTGAAAAGTAAGCAAGTTTATTTGACATATGAAGAGCATGACAAGATTACCGCTGATACACAAGCTGTGACACATGCTGCTTTCTTAAGTATGGGATCTGCGTGGGCAAAGATAAAGATTTATCCTTGGACTCTGGGTGTAAACAAATGGTACGGTGGCCTAGAAAATGTGAAAGTTAATATATCACTAAGAATCTATTCGAACAAGTGGCATGTTTACGCAGGATTAGCCATAACAAACCCAAGTGCACATCAGCAAATTCTTCAATATGCAACCAGTGCAACAGAACTATTTAGTTTAATGATAGATAACAAAGAACAAGAACTTACTGATAGACTATTAAAAGCTAAGCAATTTGTATTTGGAAAGCATACTGGTCTCTTACTATTGGATGACACGATTTTAGAGAAATATTCGCTATCAAAAAGCAGCATTGGTAACAGCAACAATTGCAAGCCAGTGCCGAATTCACATTTATCATTGTTGGCGATTGTTGATTCGTGGTTTCAACTTGGTATTGATCCATATGATCATATGATTTGTTCGACGCCATTATTCAGAATATTCCTGGGTGTGTCCGAATATCTTTTTTTAAAACCTGGCTTATTAGAACAGACAATTGATGCAGCTATCCATGATAAATCATTCATAAAAGATGATTTAGAATTTGTTATTTCGGCTAGAGAATGGAGCTCGGTTGTTTCTTTTGCCAATTTTGATATATACAAAAAGCAATTTCAGAGTGTTCAAAAGTTCTTTGAGCCAATGCTTCCAGAGGCTAATCTCATTGGCAACGAGATGATAAAAACCATTCTGAGTCATTCTAGTGACCGTTCGGCCGCTGAAAAAAGA AATACATAA

In some embodiments, a pdh gene of use in the methods disclosed hereinhas a nucleic acid sequence at least 70%, 75%, 85%, 90%, 95%, 96%, 97%,98% or 99% identical to the nucleic acid sequence set forth in SEQ IDNO: 1 or any of the PDH GenBank Accession Nos. disclosed herein. Nucleicacid sequences that do not show a high degree of identity maynevertheless encode similar amino acid sequences, due to the degeneracyof the genetic code. It is understood that changes in nucleic acidsequence can be made using this degeneracy to produce multiple nucleicacid sequences that each encode substantially the same protein. In someexamples, the pdh nucleic acid is codon-optimized for expression in theselected organism (such as algae, for example, Chlamydomonas).

In some examples, the pdh nucleic acid encodes a protein that includesor consists of the amino acid sequence set forth as:

(SEQ ID NO: 2) MVSEDKIEQWKATKVIGIIGLGDMGLLYANKFTDAGWGVICCDREEYYDELKEKYASAKFELVKNGHLVSRQSDYIIYSVEASNISKIVATYGPSSKVGTIVGGQTSCKLPEIEAFEKYLPKDCDIITVHSLHGPKVNTEGQPLVIINHRSQYPESFEFVNSVMACLKSKQVYLTYEEHDKITADTQAVTHAAFLSMGSAWAKIKIYPWTLGVNKWYGGLENVKVNISLRIYSNKWHVYAGLAITNPSAHQQILQYATSATELFSLMIDNKEQELTDRLLKAKQFVFGKHTGLLLLDDTILEKYSLSKSSIGNSNNCKPVPNSHLSLLAIVDSWFQLGIDPYDHMICSTPLFRIFLGVSEYLFLKPGLLEQTIDAAIHDKSFIKDDLEFVISAREWSSVVSFANFDIYKKQFQSVQKFFEPMLPEANLIGNEMIKTILSHSSDRSAAEKR NT

In some embodiments, the polypeptide encoded by the pdh gene has anamino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%identical to the amino acid sequence set forth in SEQ ID NO: 2 or apolypeptide encoded by any of the PDH GenBank Accession Nos. disclosedherein.

Exemplary nucleic acid and amino acid sequences can be obtained usingcomputer programs that are readily available on the internet and theamino acid sequences set forth herein. In one example, the PDHpolypeptide retains a function of the PDH protein, such as catalyzingconversion of prephenate to 4-hydroxyphenylpyruvate. Thus, a specific,non-limiting example of a PDH polypeptide is a conservative variant ofthe PDH polypeptide (such as a single conservative amino acidsubstitution, for example, one or more conservative amino acidsubstitutions, for example 1-10 conservative substitutions, 2-5conservative substitutions, 4-9 conservative substitutions, such as 1,2, 5 or 10 conservative substitutions). A table of conservativesubstitutions is provided above (Table 1).

B. Homogentisate Solanesyl Transferase (HST)

Specific disclosed transgenic cells express a heterologous hst nucleicacid. The hst nucleic acid (such as all or a portion of an hst gene)encodes a protein capable of catalyzing condensation of homogentisateand solanesyl diphosphate to form 2-demthylplastoquinol-9. In someexamples, the hst nucleic acid or protein is an Arabidopsis thaliana hstnucleic acid or protein. Nucleic acid and amino acid sequences for hstare publicly available. For example, GenBank Accession Nos. NM_001084669and NM_001161137 disclose exemplary A. thaliana hst nucleic acidsequences. GenBank Accession Nos. NP_001154609 and NP_001078138 discloseexemplary A. thaliana HST amino acid sequences. In other examples, theHST nucleic acid or protein is from a plant, such as Arabidopsis, corn,rice, or spinach, or an algae, such as Chlamydomonas (see e.g., Sadre etal., J. Biol. Chem. 285:18191-18198, 2010). Exemplary hst nucleic acidsequences include GenBank Accession Nos. NM_001066618 (Oryza sativa),NM_001153231 (Zea mays), and XM_001695289 (Chlamydomonas reinhardtii),each of which is incorporated herein by reference. One of ordinary skillin the art can identify additional HST nucleic acid and amino acidsequences, for example, HST sequences from other organisms.

In one non-limiting example, an hst nucleic acid is from A. thaliana. Insome examples, the hst nucleic acid includes or consists of the nucleicacid sequence set forth as:

(SEQ ID NO: 3) ATGTGTTCTCAGGTTGGTGCTGCTGAGTCTGATGATCCAGTGCTGGATAGAATTGCCCGGTTCCAAAATGCTTGCTGGAGATTTCTTAGACCCCATACAATCCGCGGAACAGCTTTAGGATCCACTGCCTTGGTGACAAGAGCTTTGATAGAGAACACTCATTTGATCAAATGGAGTCTTGTACTAAAGGCACTTTCAGGTCTTCTTGCTCTTATTTGTGGGAATGGTTATATAGTCGGCATCAATCAGATCTACGACATTGGAATCGACAAAGTGAACAAACCATACTTGCCAATAGCAGCAGGAGATCTATCAGTGCAGTCTGCTTGGTTGTTAGTGATATTTTTTGCGATAGCAGGGCTTTTAGTTGTCGGATTTAACTTTGGTCCATTCATTACAAGCCTATACTCTCTTGGCCTTTTTCTGGGAACCATCTATTCTGTTCCACCCCTCAGAATGAAAAGATTCCCAGTTGCAGCATTTCTTATTATTGCCACGGTACGAGGTTTCCTTCTTAACTTTGGTGTGTACCATGCTACAAGAGCTGCTCTTGGACTTCCATTTCAGTGGAGTGCACCTGTGGCGTTCATCACATCTTTTGTGACACTGTTTGCACTGGTCATTGCTATTACAAAGGACCTTCCTGATGTTGAAGGAGATCGAAAGTTCCAAATATCAACCCTGGCAACAAAACTTGGAGTGAGAAACATTGCATTCCTCGGTTCTGGACTTCTGCTAGTAAATTATGTTTCAGCCATATCACTAGCTTTCTACATGCCTCAGGTTTTTAGAGGTAGCTTGATGATTCCTGCACATGTGATCTTGGCTTCAGGCTTAATTTTCCAGACATGGGTACTAGAAAAAGCAAACTACACCAAGGAAGCTATCTCAGGATATTATCGGTTTATATGGAATCTCTTCTACGCAGAGTATCTGTTATTCCCCTTCCTCTAGCTTTCAATTTCATGGTGAGGATATGCAGTTTTCTTTGTATATCATTCTTCTTCTTCTTTGTAGCTTGGAGTCAAAATCGGTTCCTTCATGTACATACATCAAGGATATGTCCTTCTGAGCA

In some embodiments, an hst nucleic acid of use in the methods disclosedherein has a nucleic acid sequence at least 70%, 75%, 85%, 90%, 95%,96%, 97%, 98% or 99% identical to the nucleic acid sequence set forth inSEQ ID NO: 3 or any of the HST GenBank Accession Nos. disclosed herein.Nucleic acid sequences that do not show a high degree of identity maynevertheless encode similar amino acid sequences, due to the degeneracyof the genetic code. It is understood that changes in nucleic acidsequence can be made using this degeneracy to produce multiple nucleicacid sequences that each encode substantially the same protein. In someexamples, the hst nucleic acid is codon-optimized for expression in theselected organism, (such as algae, for example, Chlamydomonas).

In some examples, the hst nucleic acid encodes a protein that includesor consists of the amino acid sequence set forth as:

(SEQ ID NO: 4) MCSQVGAAESDDPVLDRIARFQNACWRFLRPHTIRGTALGSTALVTRALIENTHLIKWSLVLKALSGLLALICGNGYIVGINQIYDIGIDKVNKPYLPIAAGDLSVQSAWLLVIFFAIAGLLVVGFNFGPFITSLYSLGLFLGTIYSVPPLRMKRFPVAAFLIIATVRGFLLNFGVYHATRAALGLPFQWSAPVAFITSFVTLFALVIAITKDLPDVEGDRKFQISTLATKLGVRNIAFLGSGLLLVNYVSAISLAFYMPQVFRGSLMIPAHVILASGLIFQTWVLEKANYTKEAISGYY RFIWNLFYAEYLLFPFL

In some embodiments, the polypeptide encoded by the hst nucleic acid hasan amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%identical to the amino acid sequence set forth in SEQ ID NO: 4 or any ofthe HST GenBank Accession Nos. disclosed herein.

Exemplary nucleic acid and amino acid sequences can be obtained usingcomputer programs that are readily available on the internet and theamino acid sequences set forth herein. In one example, the HSTpolypeptide retains a function of the HST protein, such as condensationof homogentisate and solanesyl diphosphate to form2-demthylplastoquinol-9. Thus, a specific, non-limiting example of anHST polypeptide is a conservative variant of the HST polypeptide (suchas a single conservative amino acid substitution, for example, one ormore conservative amino acid substitutions, for example 1-10conservative substitutions, 2-5 conservative substitutions, 4-9conservative substitutions, such as 1, 2, 5 or 10 conservativesubstitutions). In one example, an HST polypeptide is a conservativevariant of SEQ ID NO: 4, for example including one or more conservativeamino acid substitutions (for example 1-10 conservative substitutions,2-5 conservative substitutions, 4-9 conservative substitutions, such as1, 2, 5 or 10 conservative substitutions). A table of conservativesubstitutions is provided above (Table 1).

C. Deoxyxylulose Phosphate Synthase (DXS)

Specific transgenic cells disclosed herein express a heterologous dxsnucleic acid in an algae cell. The dxs nucleic acid (such as all or aportion of a dxs gene) encodes a protein capable of catalyzing thethiamin diphosphate-dependent condensation of pyruvate andglyceraldehyde-3-phosphate to yield 1-deoxy-xylulose 5-phosphate.Nucleic acid and amino acid sequences for dxs are publicly available. Insome examples, the DXS nucleic acid or protein is from a bacteria, suchas E. coli. For example, GenBank Accession No. NC_000913.2 (437539 . . .439401, complement) discloses an exemplary E. coli dxs nucleic acidsequence. GenBank Accession No. NP_414954 discloses an exemplary E. coliDXS amino acid sequence. In other examples, the DXS nucleic acid orprotein is from a plant, such as Arabidopsis, corn, tomato, pepper,spinach, potato, gingko, or cassaya, or an algae, such as Chlamydomonas(see e.g., Cordoba et al., J. Exp. Bot. 62:2023-2038, 2011; Sayre etal., Ann. Rev. Plant Biol. 62:251-272, 2011). Exemplary dxs nucleic acidsequences include GenBank Accession Nos. NM_117647 (Arabidopsisthaliana), NM_001247743 (Solanum lycopersicum), NM_001288201 (Solanumtuberosum), and XM_001702010 (Chlamydomonas reinhardtii) each of whichis incorporated herein by reference. One of ordinary skill in the artcan identify additional DXS nucleic acid and amino acid sequences, forexample, DXS sequences from other organisms.

Exemplary nucleic acid and amino acid sequences can be obtained usingcomputer programs that are readily available on the internet and theamino acid sequences set forth herein. In one example, the DXSpolypeptide retains a function of the DXS protein, such as condensationof condensation of pyruvate and glyceraldehyde-3-phosphate to yield1-deoxy-xylulose 5-phosphate. Thus, a specific, non-limiting example ofa DXS polypeptide is a conservative variant of the DXS polypeptide (suchas a single conservative amino acid substitution, for example, one ormore conservative amino acid substitutions, for example 1-10conservative substitutions, 2-5 conservative substitutions, 4-9conservative substitutions, such as 1, 2, 5 or 10 conservativesubstitutions). A table of conservative substitutions is provided above(Table 1).

D. Production of Transgenic Algae Cells

Methods of transforming and cultivating algae are known in the art(e.g., Stern et al., The Chlamydomonas Sourcebook: A Comprehensive Guideto Biology and Laboratory Use, Second Edition, Academic Press, 2008). Inan embodiment, a nucleic acid molecule can be inserted into one or moreexpression vectors, using methods known to those of skill in the art.Vectors include one or more expression cassettes including expressioncontrol sequences operably linked to the nucleic acid of interest (suchas pdh, hst, dxs, or other nucleic acids). An expression cassetteincludes nucleic acid elements that permit expression of a gene or othernucleic acid in a host cell. Vectors are discussed in more detail below.

Transformation of an alga cell with recombinant DNA can be carried outby conventional techniques as are well known to those of ordinary skillin the art. Methods of transformation include transformation utilizingAgrobacterium tumifaciens transformed with a plasmid including thedesired nucleic acid. In other examples, algae cells can be transformedutilizing biolistics (e.g., the “gene gun”), electroporation, glassbeads, or carbide whiskers. One of ordinary skill in the art can selectan appropriate transformation method and vector, based on the cells tobe transformed and other desired characteristics.

In some embodiments, the cells are transformed with two or moreheterologous nucleic acids. In some examples, each heterologous nucleicacid is separately introduced to the cell, for example in separatetransformation vectors. The cells can be transformed with the separatevectors sequentially or simultaneously. In other examples, the two ormore heterologous nucleic acids are introduced to the cell in the sametransformation vector under the control of the same promoter (forexample, a bi-cistronic construct) or under the control of separatepromoters. One of ordinary skill in the art can select appropriatevectors and methods to transform cells with two or more heterologousnucleic acids.

In some examples, the heterologous nucleic acid (such as a heterologousnucleic acid encoding a PDH protein, an HST protein, or a DXS protein)is codon-optimized for the cell in which it is to be expressed. Codonusage bias, the use of synonymous codons at unequal frequencies, isubiquitous among genetic systems (Ikemura, J. Mol. Biol. 146:1-21, 1981;Ikemura, J. Mol. Biol. 158:573-97, 1982). The strength and direction ofcodon usage bias is related to genomic G+C content and the relativeabundance of different isoaccepting tRNAs (Akashi, Curr. Opin. Genet.Dev. 11:660-666, 2001; Duret, Curr. Opin. Genet. Dev. 12:640-9, 2002;Osawa et al., Microbiol. Rev. 56:229-264, 1992). Codon usage can affectthe efficiency of gene expression. Codon-optimization refers toreplacement of a codon in a nucleic acid sequence with a synonymouscodon (one that codes for the same amino acid) more frequently used(preferred) in the organism. Each organism has a particular codon usagebias for each amino acid, which can be determined from publiclyavailable codon usage tables (for example see Nakamura et al., NucleicAcids Res. 28:292, 2000 and references cited therein). For example, acodon usage database is available on the World Wide Web atwww.kazusa.or.jp/codon. One of ordinary skill in the art can modify anucleic acid encoding a particular amino acid sequence, such that itencodes the same amino acid sequence, while being optimized forexpression in a particular cell type (such as an algae cell).

A wide variety of algae species (such as microalgae and/or macroalgae)can be utilized in the methods described herein. In some examples, thealgae species include, but are not limited to Chlorella (such asChlorella vulgaris), Chlamydomonas (such as Chlamydomonas reinhardtii),Chaetoceros, Spirulina (such as Spirulina platensis), Dunaliella, andPorphyridum. In particular examples, the algae species include algaeuseful for production of biofuels or other compounds (such aspolyunsaturated acids, pigments, or phytochemicals, for example, fornutritional supplements). In some examples, the algae includeAkistrodesmus, Arthrospira, Botryococcus braunii, Chlorella (such asChlorella sp. or Chlorella protothecoides), Crypthecodinium (such asCrypthecodinium cohnii), Cyclotella, Dunaliella tertiolecta, Galdieria(such as Galdieria sulphuraria), Gracilaria, Hantzschia, Haematococcus(such as Haematococcus pluvialis), Nannochloris, Nannochloropsis,Neochloris oleoabundans, Nitzschia, Phaeodactylum, Pleurochrysiscarterae (also called CCMP647), Porphyridium, Sargassum, Scenedesmus(such as Scenedesmus obliquus), Schiochytrium, Stichococcus,Tetraselmis, Thalassiosira pseudonana, Thraustochytrium roseum, andUlkenia sp. In one example, the algae species is Chlamydomonasreinhardtii.

E. Production of Transgenic Plant Cells

Transformation and regeneration of both monocotyledonous anddicotyledonous plant cells is routine, and the most appropriatetransformation technique will be determined by the practitioner. Thechoice of method will vary with the type of plant to be transformed;those skilled in the art will recognize the suitability of particularmethods for given plant types. Suitable methods may include, but are notlimited to: electroporation of plant protoplasts; liposome-mediatedtransformation; polyethylene glycol (PEG)-mediated transformation;transformation using viruses; micro-injection of plant cells;micro-projectile bombardment of plant cells; vacuum infiltration; andAgrobacterium tumefaciens (AT) mediated transformation.

A number of vectors suitable for stable transformation of plant cells orfor the establishment of transgenic plants have been described in, e.g.,Pouwels et al. (Cloning Vectors: A Laboratory Manual, 1985, Suppl.,1987), Weissbach and Weissbach (Meth. Plant Mol. Bio., Academic Press,1989) and Gelvin et al. (Plant Molecular Biology Manual, Kluwer AcademicPublishers, 1990). In addition, one of ordinary skill in the art isaware of the components useful in a transformation vector, and will beable to select and assemble such components in order to tailor make avector for their specific use. Additional vectors are discussed below.

In some embodiments, the cells are transformed with two or moreheterologous nucleic acids. In some examples, each heterologous nucleicacid is separately introduced to the cell, for example in separatetransformation vectors. The cells can be transformed with the separatevectors sequentially or simultaneously. In other examples, the two ormore heterologous nucleic acids are introduced to the cell in the sametransformation vector under the control of the same promoter (forexample, a bi-cistronic construct) or under the control of separatepromoters. One of ordinary skill in the art can select appropriatevectors and methods to transform cells with two or more heterologousnucleic acids.

Numerous methods for transforming plant cells with recombinant DNA areknown in the art and may be used. Two commonly used methods for planttransformation are Agrobacterium-mediated transformation andmicroprojectile bombardment. Microprojectile bombardment methods (e.g.,the “gene gun”) are illustrated in U.S. Pat. No. 5,015,580 (soybean);U.S. Pat. No. 5,550,318 (corn); U.S. Pat. No. 5,538,880 (corn); U.S.Pat. No. 5,914,451 (soybean); U.S. Pat. No. 6,160,208 (corn); U.S. Pat.No. 6,399,861 (corn) and U.S. Pat. No. 6,153,812 (wheat) andAgrobacterium-mediated transformation is described in U.S. Pat. No.5,159,135 (cotton); U.S. Pat. No. 5,824,877 (soybean); U.S. Pat. No.5,591,616 (corn); and U.S. Pat. No. 6,384,301 (soybean), all of whichare incorporated herein by reference. For Agrobacterium tumefaciensbased plant transformation system, additional elements present ontransformation constructs will include T-DNA left and right bordersequences to facilitate incorporation of the recombinant polynucleotideinto the plant genome.

Transformation methods are preferably practiced in tissue culture onmedia and in a controlled environment. “Media” refers to the numerousnutrient mixtures that are used to grow cells in vitro, that is, outsideof the intact living organism. Recipient cell targets include, but arenot limited to, meristem cells, callus, immature embryos and gameticcells such as microspores, pollen, sperm and egg cells. It iscontemplated that any cell from which a fertile plant may be regeneratedis useful as a recipient cell. Callus may be initiated from tissuesources including, but not limited to, immature embryos, seedling apicalmeristems, microspores and the like. Cells capable of proliferating ascallus are also recipient cells for genetic transformation. Practicaltransformation methods and materials for making transgenic plants, e.g.various media and recipient target cells, transformation of immatureembryos and subsequent regeneration of fertile transgenic plants aredisclosed in U.S. Pat. Nos. 6,194,636 and 6,232,526, which areincorporated herein by reference.

Following transformation and regeneration of plants with thetransformation vector, transformed plants may be selected using adominant selectable marker incorporated into the transformation vector.Typically, such a marker will confer antibiotic resistance on theseedlings of transformed plants, and selection of transformants can beaccomplished by exposing the seedlings to appropriate concentrations ofthe antibiotic. After transformed plants are selected and grown tomaturity, they can be assayed using the methods described herein, andother methods appropriate to the synthetic construct of the transgene,to determine whether the introduced nucleic acid(s) are being produced.

The seeds of transgenic plants can be harvested from fertile transgenicplants and be used to grow progeny generations of transformed plantsincluding hybrid plant lines for screening of plants having an enhancedagronomic trait. In addition to direct transformation of a plant with arecombinant DNA, transgenic plants can be prepared by crossing a firstplant having a recombinant DNA with a second plant lacking the DNA. Forexample, recombinant DNA can be introduced into first plant line that isamenable to transformation to produce a transgenic plant which can becrossed with a second plant line to introgress the recombinant DNA intothe second plant line. A transgenic plant with recombinant DNA providingan enhanced agronomic trait, e.g. enhanced yield, can be crossed withtransgenic plant line having other recombinant DNA that confers anothertrait, e.g. herbicide resistance or pest resistance, to produce progenyplants having recombinant DNA that confers both traits. Typically, insuch breeding for combining traits the transgenic plant donating theadditional trait is a male line and the transgenic plant carrying thebase traits is the female line. The progeny of this cross will segregatesuch that some of the plants will carry the DNA for both parental traitsand some will carry DNA for one parental trait; such plants can beidentified by markers associated with parental recombinant DNA Progenyplants carrying DNA for both parental traits can be crossed back intothe female parent line multiple times, e.g. usually 6 to 8 generations,to produce a progeny plant with substantially the same genotype as oneoriginal transgenic parental line, but for the recombinant DNA of theother transgenic parental line

In the practice of transformation DNA is typically introduced into onlya small percentage of target cells in any one transformation experiment.Marker genes are used to provide an efficient system for identificationof those cells that are stably transformed by receiving and integratinga transgenic DNA construct into their genomes. Preferred marker genesprovide selective markers which confer resistance to a selective agent,such as an antibiotic or herbicide. Any of the herbicides to whichplants may be resistant are useful agents for selective markers.Potentially transformed cells are exposed to the selective agent. In thepopulation of surviving cells will be those cells where, generally, theresistance-conferring gene is integrated and expressed at sufficientlevels to permit cell survival. Cells may be tested further to confirmstable integration of the exogenous DNA. Commonly used selective markergenes include those conferring resistance to antibiotics such askanamycin and paromomycin (val), hygromycin B (aph IV) and gentamycin(aac3 and aacC4) or resistance to herbicides such as glufosinate (bar orpat) and glyphosate (aroA or EPSPS). Examples of such selectable areillustrated in U.S. Pat. Nos. 5,550,318; 5,633,435; 5,780,708 and6,118,047, all of which are incorporated herein by reference. Screenablemarkers which provide an ability to visually identify transformants canalso be employed, e.g., a gene expressing a colored or fluorescentprotein such as a luciferase or green fluorescent protein (GFP) or agene expressing a beta-glucuronidase or uidA gene (GUS) for whichvarious chromogenic substrates are known.

Cells that survive exposure to the selective agent, or cells that havebeen scored positive in a screening assay, may be cultured inregeneration media and allowed to mature into plants. Developingplantlets can be transferred to plant growth mix, and hardened off,e.g., in an environmentally controlled chamber at about 85% relativehumidity, 600 ppm CO₂, and 25-250 microeinsteins m⁻² s⁻¹ of light, priorto transfer to a greenhouse or growth chamber for maturation. Plants areregenerated from about 6 weeks to 10 months after a transformant isidentified, depending on the initial tissue. Plants may be pollinatedusing conventional plant breeding methods known to those of skill in theart and seed produced, e.g. self-pollination is commonly used withtransgenic corn. The regenerated transformed plant or its progeny seedor plants can be tested for expression of the recombinant DNA andscreened for the presence of enhanced agronomic trait.

In some examples, callus and suspension cultures can be established fromthe disclosed transgenic plant cells by protocols known in the art.Suspension cultures can be raised from a callus culture and maintainedin fresh suspension medium. Suitable nutrient media for plant cellsuspension culture are well known to one of skill in the art.

In a particular example, a plant cell suspension medium includesMurashige and Skoog (MS) salts (e.g., Cat. No. M524, Phytotech, ShawneeMission, Kans.) and Nitsch and Nitsch vitamins (e.g., Cat. No. N608;Phytotech, Shawnee Mission, Kans.). See, e.g., Nitsch and Nitsch,Science 163:85-87, 1969. Suspension cultures can be established byaseptically transferring a known mass of cells expressed as packed cellvolume (PCV) to fresh medium on a regular schedule, typically at 7-14day intervals. Medium for suspension culture (“suspension medium”) canbe optimized for initiation of suspension culture or for desiredcharacteristics.

Representative, non-limiting example plant cells that can be used in themethods described herein include Arabidopsis; field crops (e.g. alfalfa,barley, bean, clover, corn, cotton, flax, false flax (Camelina),lentils, maize, oats, pea, rape/canola, rice, rye, safflower, sorghum,soybean, sugarcane, sunflower, tobacco, and wheat); vegetable crops(e.g. asparagus, beet, brassica generally, broccoli, Brussels sprouts,cabbage, carrot, cauliflower, celery, cucumber (cucurbits), eggplant,lettuce, mustard, onion, pepper, potato, pumpkin, radish, spinach,squash, taro, tomato, and zucchini); fruit and nut crops (e.g. almond,apple, apricot, banana, blackberry, blueberry, cacao, cassaya, cherry,citrus, coconut, cranberry, date, hazelnut, grape, grapefruit, guava,kiwi, lemon, lime, mango, melon, nectarine, orange, papaya, passionfruit, peach, peanut, pear, pineapple, pistachio, plum, raspberry,strawberry, tangerine, walnut, and watermelon); tree woods andornamentals (e.g. alder, ash, aspen, azalea, birch, boxwood, camellia,carnation, chrysanthemum, elm, fir, grasses (such as switch grass orMiscanthus), ivy, jasmine, juniper, oak, palm, poplar, pine, redwood,rhododendron, rose, and rubber).

F. Transformation and Expression Constructs

One of ordinary skill in the art can select or construct vectors fortransformation and expression of heterologous nucleic acids in cells(such as plant or algae cells). Typically, transformation and expressionvectors include, for example, one or more nucleic acids under thetranscriptional control of 5′ and 3′ regulatory sequences and a dominantselectable marker. For example, genes that confer antibiotic resistanceor sensitivity to the plasmid may be used as selectable markers. Suchexpression vectors also can contain a promoter regulatory region (e.g.,a regulatory region controlling inducible or constitutive,environmentally-or developmentally-regulated, or cell- ortissue-specific expression), a transcription initiation start site, aribosome binding site, an RNA processing signal, a transcriptiontermination site, and/or a polyadenylation signal.

In some embodiments, the promoters selected for vectors of use in thedisclosed methods are chloroplast promoters. In one example, the nucleicacid is placed under the control of the strong constitutive promoter(for example an alpha subunit of ATP synthase (atpA) promoter).Additional promoters suitable for use in chloroplast gene expressioninclude the 16S RNA promoter, the beta subunit of the ATP synthase(atpB) promoter, the ribulose-1,5-bisphosphate carboxylase/oxygenaselarge subunit (rbcL) promoter, the photosystem II D1 protein (psbA),promoter, the ribulose bisphosphate carboxylase (RBCS2) promoter, or theHSP70A-RBCS2 tandem promoter.

In some examples, a strong, medium, or weak strength promoter may beselected to tune the amount of transgenic protein produced, and thusproduce transgenic cell lines with varying PQ pool levels. Promoterstrength can be determined by measuring rates of transcription from apromoter, for example in an in vitro or in vivo system. In certainembodiments, a strong promoter is one which promotes transcription ofRNA at high levels, for example at levels such that the transcriptionalactivity of the promoter generally accounts for about 5% or more of thetranscriptional activity of all transcription within a cell. Thestrength of a promoter is often cell or tissue-specific and thus mayvary from one cell type to another. In some examples, the rbcL, psbA,and atpB promoters are strong promoters, while the atpA promoter is aweaker promoter. See e.g., Blowers et al., Plant Cell 2:1059-1070, 1990;Hwang et al., Proc. Natl. Acad. Sci. USA 93:996-1000, 1996; Salvador etal., Plant J. 3:213-219, 1993; Silk and Wu, Plant Mol. Biol. 23:87-96,1993.

In additional embodiments, the nucleic acids disclosed herein areintroduced into the nuclear genome (for example, in a vector including anuclear gene promoter). In such examples, the expression vector alsoincludes a chloroplast targeting sequence operably linked to the 5′ endof the nucleic acid to be expressed. Examples of chloroplast targetingsequences (for example, sequences encoding chloroplast “transitpeptides”) are known to one of ordinary skill in the art, and includethose from rbcS, Cab, DnaJ-J8, biotin carboxyl carrier protein (BCCP),protochlorophyllide oxidoreductase A (PORA), ferredoxin-dependentglutamate synthase 2 (GLU2), tocopherol cyclase (TOCC), and most Calvincycle enzymes (e.g., Rubisco small subunit, phosphoglycerate kinase, andglyceraldehyde 3-phosphate dehydrogenase). See, e.g., Lee et al., PlantCell 20:1603-1622, 2008; Jang et al., Mol. Breeding. 9:81-91, 2002;incorporated herein by reference in their entirety.

Examples of additional promoters that can be used in the presentdisclosure include, but are not limited to the Cauliflower mosaic virus35S promoter, SV40 promoter, the CMV enhancer-promoter, the CMVenhancer/β-actin promoter, and the tissue-specific promoter probasin.Other promoter sequences that can be used to construct nucleic acids andpractice methods disclosed herein include, but are not limited to: thelac system, the trp system, the tac system, the trc system, majoroperator and promoter regions of phage lambda, the control region of fdcoat protein, the early and late promoters of SV40, promoters derivedfrom polyoma, adenovirus, retrovirus, baculovirus and simian virus, thepromoter for 3-phosphoglycerate kinase, the promoters of yeast acidphosphatase, the promoter of the yeast alpha-mating factors, anyretroviral LTR promoter such as the RSV promoter; inducible promoters,such as the MMTV promoter; the metallothionein promoter; heat shockpromoters; the albumin promoter; the histone promoter; the α-actinpromoter; TK promoters; B19 parvovirus promoters; the SV10 latepromoter; the ApoAI promoter and combinations thereof.

Examples of additional strong promoters include, but are not limited to:viral promoters (such as CaMV 35S or CoYMV), ubiquitin promoter (such asUbi-1 from maize), actin promoter (e.g, Act from rice), atpA promoter,nopaline synthase promoter, and the octopine synthase promoter, pEMUpromoter, MAS promoter, or a H3 histone promoter.

Inducible promoters or gene-switches are used to both spatially andtemporally regulate gene expression. By allowing the time and/orlocation of gene expression to be precisely regulated, gene-switches orinducible promoters may control deleterious and/or abnormal effectscaused by overexpression or non-localized gene expression. Thus, for atypical inducible promoter in the absence of the inducer, there would belittle or no gene expression while, in the presence of the inducer,expression should be high (e.g., off/on). Examples ofstimulus-responsive promoters include, but are not limited tohormone-responsive promoters (e.g, ethanol inducible alcR-encodedtranscriptional activator (ALCR), a promoter derived from alcA),light-inducible promoters (such as rbcS promoter, Cab promoter),metal-inducible promoters, heat-shock promoters, wound-inducible andstress-inducible (e.g., drought stress, salt stress, shear stress,nutrient stress) promoters. Others are activated by chemical stimuli,such as IPTG or Tetracycline (Tet), or galactose. Other promoters areresponsive to pathogen infection or insect damage.

A number of controllable gene expression systems have been devised,including those regulated by light (e.g., the pea rbcS-3A promoter,Kuhlemeier et al., The Plant Cell, 1:471-478, 1989, and the maize rbcSpromoter, Schaffner and Sheen, Plant Cell 3:997, 1991), heat (Callis etal., Plant Physiol. 88:965, 1988; Ainley and Key, Plant Mol. Biol.,14:949-967, 1990; Holtorf et al., Plant Mol. Biol. 29:637-646, 1995),pathogens (PR1-a; Williams et al., Biotechnology 10:540-543, 1992; Gatz,Annu. Rev. Plant Physiol. Plant Mol. Biol. 48:89-108, 1997), herbicidesafeners (In2-2, GST-27; De Veylder et al., Plant Cell Physiol.38:568-577, 1997), wounding (Firek et al. Plant Mol. Biol. 22:129-212,1993), ethanol (Salter et al., Plant J. 16:127-132, 1998), phytohormones(Li et al., Plant Cell 3:1167-1175, 1991), steroids (Aoyama and Chua,Plant J., 11:605-612, 1997), wounding (e.g., wunI, Siebertz et al.,Plant Cell 1:961, 1989), hormones, such as abscisic acid (Marcotte etal., Plant Cell 1:969, 1989); chemicals such as methyl jasminate orsalicylic acid (see Gatz et al., Ann. Rev. Plant Physiol. Plant Mol.Biol. 48:89-108 1997), and tetracycline (Gatz et al., Plant J.2:397-404, 1992; Weinmann et al., Plant J., 5:559-569, 1994; Sommer etal., Plant Cell Rep. 17:891-896, 1998) (from Granger & Cyr, Plant CellReports 20:227-234, 2001).

In another embodiment, a promoter is a tissue-specific, cell-specific,or developmental stage-specific promoter, which promotes transcriptionin a single cell or tissue type, a narrow range of cells or tissues, orin one or more specific developmental stages, or at least promotesmeasurable more transcription in such. Examples of such promotersinclude, but are not limited to: anther-specific, embryo-specific,endosperm-specific, floral-specific, leaf-specific, meristem-specific,nodule-specific, phloem-specific, seed-specific, stem-specific,stomata-specific, trichome-specific, root-specific, tapetum-specific,and xylem-specific promoters. See, for instance, Carpenter et al., ThePlant Cell 4:557-571, 1992, Denis et al., Plant Physiol. 101:1295-13041993, Opperman et al., Science 263:221-223, 1993, Stockhause et al., ThePlant Cell 9:479-489, 1997; Roshal et al., EMBO J. 6:1155, 1987;Schernthaner et al., EMBO J. 7:1249, 1988; and Bustos et al., Plant Cell1:839, 1989.

It is specifically contemplated that useful promoters will includepromoters present in plant or algae genomes as well as promoters fromother sources, including nopaline synthase (nos) promoter and octopinesynthase (ocs) promoters carried on tumor-inducing plasmids ofAgrobacterium tumefaciens, caulimovirus promoters such as thecauliflower mosaic virus or figwort mosaic virus promoters. Forinstance, see U.S. Pat. Nos. 5,322,938 and 5,858,742 which discloseversions of the constitutive promoter derived from cauliflower mosaicvirus (CaMV35S), U.S. Pat. No. 5,378,619 which discloses a FigwortMosaic Virus (FMV) 35S promoter, U.S. Pat. No. 5,420,034 which disclosesa napin promoter, U.S. Pat. No. 6,437,217 which discloses a maize RS81promoter, U.S. Pat. No. 5,641,876 which discloses a rice actin promoter,U.S. Pat. No. 6,426,446 which discloses a maize RS324 promoter, U.S.Pat. No. 6,429,362 which discloses a maize PR-1 promoter, U.S. Pat. No.6,232,526 which discloses a maize A3 promoter, U.S. Pat. No. 6,177,611which discloses constitutive maize promoters, U.S. Pat. No. 6,433,252which discloses a maize L3 oleosin promoter, U.S. Pat. No. 6,429,357which discloses a rice actin 2 promoter and intron, U.S. Pat. No.5,837,848 which discloses a root specific promoter, U.S. Pat. No.6,084,089 which discloses cold inducible promoters, U.S. Pat. No.6,294,714 which discloses light inducible promoters, U.S. Pat. No.6,140,078 which discloses salt inducible promoters, U.S. Pat. No.6,252,138 which discloses pathogen inducible promoters, U.S. Pat. No.6,175,060 which discloses phosphorus deficiency inducible promoters,U.S. Pat. No. 6,635,806 which discloses a coixin promoter, U.S.2002/0192813 A1 which discloses 5′, 3′ and intron elements useful in thedesign of effective plant expression vectors, U.S. 2004/0216189 A1 whichdiscloses a maize chloroplast aldolase promoter, and U.S. 2004/0123347A1 which discloses water-deficit inducible promoters, all of which areincorporated herein by reference. These and numerous other promotersthat function in plant and algae cells are known to those skilled in theart and available for use in recombinant polynucleotides of the presentdisclosure to provide for expression of desired nucleic acids intransgenic plant or algae cells.

In one example, a specific transformation and expression vector foralgae is a pBA155 plasmid including the desired heterologous nucleicacid(s) (e.g., Minagawa and Crofts, Photosynth. Res. 42:121-131, 1994).Additional vectors for transforming algae, such as additionalchloroplast transformation vectors are known to one of ordinary skill inthe art. These include pD1, pD1-SAA, and pAtpA (Rasala and Mayfield,Bioengineered Bugs 2:50-54, 2010), inducible chloroplast expressionvectors (e.g., Surzycki et al., Proc. Natl. Acad. Sci. USA104:17548-17553, 2007), and others that can be identified by one ofordinary skill in the art.

IV. Methods of Increasing Plastoquinone Levels in Transgenic Cells

Also disclosed herein are methods of producing a photosynthetic cell(such as a plant or alga cell) with an increased amount of PQ comparedto a control cell. In some examples, the methods include expressing incell a heterologous nucleic acid encoding a PDH protein, a heterologousnucleic acid encoding an HST protein, a heterologous nucleic acidencoding a DXS protein, or a combination of two or more thereof. In someembodiments, the transgenic cells express a heterologous nucleic acidencoding a PDH protein. In other embodiments, the transgenic cellsexpress a heterologous nucleic acid encoding an HST protein. Inadditional embodiments, the transgenic cells express a heterologousnucleic acid encoding a DXS protein. In still further embodiments, thetransgenic cells express a heterologous nucleic acid encoding a PDHprotein and a heterologous nucleic acid encoding an HST protein.

In some embodiments, the method produces transgenic cells having atleast about 5% higher PQ levels (such as at least about 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500%, ormore) as compared to control cells (such as a wild type (non-transgenic)cell of the same strain or species as the transgenic cell or atransgenic cell that does not express the heterologous PDH, HST, and/orDXS protein). For example the method may produce transgenic cells havingabout 5-500% more PQ (such as about 10-400%, 20-300%, 50-250%, 75-200%,50-150%, 10-100%, or 10-50% more) than a control cell. Methods ofdetermining the amount (quantitatively or relative to a control) of PQin a cell (such as a cell or a population of cells) are known to one ofordinary skill in the art. These methods include direct measurements(for example, high pressure liquid chromatography or mass spectrometry)or indirect measurements (such as chlorophyll fluorescence or NPQ). Insome examples, the methods described in Example 2, below are used todetermine an amount of PQ (such as an increase or decrease in PQcompared to a control).

V. Methods of Increasing Biomass Production

Disclosed herein are methods of increasing photosynthetic cell biomassproduction, for example by cultivating one or more of the transgeniccells (such as a transgenic plant or alga cell) disclosed herein. Insome embodiments, the transgenic cells express a heterologous nucleicacid encoding a PDH protein. In other embodiments, the transgenic cellsexpress a heterologous nucleic acid encoding an HST protein. Inadditional embodiments, the transgenic cells express a heterologousnucleic acid encoding a DXS protein. In still further embodiments, thetransgenic cells express a heterologous nucleic acid encoding a PDHprotein and a heterologous nucleic acid encoding an HST protein.

In some embodiments, cultivating the transgenic cells disclosed hereinproduces at least about 1.2-fold more biomass (such as at least about1.3-fold, 1.4-fold, 1.5-fold, 1.75-fold, 2-fold, 2.25-fold, 2.5-fold,2.75-fold, 3-fold, 3.25-fold, 3.5-fold, 3.75-fold, 4-fold, 4.25-fold,4.5-fold, 4.75-fold, 5-fold, 5.25-fold, 5.5-fold, 5.75-fold, 6-fold,7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, or more) as comparedto control cells cultivated under the same growth conditions (such aswild type (non-transgenic) cells of the same strain or species as thetransgenic cells or transgenic cells that do not express theheterologous PDH, HST, and/or DXS protein). For example the transgeniccells may produce about 1.2-50-fold more biomass (such as about2-20-fold, about 3-10-fold, about 2-10-fold, about 3-6-fold, or about1-5-fold more biomass) than control cells. The amount of biomass in aculture can be determined by the number of cells present, wet weight,dry weight, amount of a cellular constituent (such as chlorophyll-a),absorbance (e.g., OD₇₅₀), or any other measurement known to one ofordinary skill in the art.

Methods of cultivating plant and algae cells are well known to one ofordinary skill in the art and include those described herein. Thetransgenic cells of the present disclosure can be cultured inconventional fermentation bioreactors, which include, but are notlimited to, batch, fed-batch, cell recycle, and continuous fermenters.The cultivation can also be conducted in shake flasks, test tubes,microtiter dishes, or petri plates. Transgenic cells can also becultivated in outdoor open ponds or raceways. Cultivation of the cellsis carried out at a temperature, pH, oxygen content, and lightconditions appropriate for the particular recombinant species. Suchculturing conditions are well within the expertise of one of ordinaryskill in the art.

In some examples, the cells are cultivated in a liquid medium. In someexamples, the cells are cultured in the liquid medium for about 12 hoursor more, for example, about 12, hours, 24 hours, 36 hours, 48 hours, 60hours, 72 hours, 84 hours, 96 hours, or more. In further examples, thecells are cultured for about 1 day to about 20 days or more, such asabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20 days, or more. In one example, the cells are cultured for at least 24hours. The cells can be grown with a light-dark cycle, such as periodsof alternating light and dark. In some examples, the cells are be grownon a standard light/dark regimen, for example alternating periods ofabout 8-16 hours of light and about 8-16 hours of dark. In someexamples, the light-dark cycle alternates periods of about 16 hours oflight with periods of about 8 hours of dark. In other examples, thelight-dark cycle alternates periods of about 12 hours of light withperiods of about 12 hours of dark. In other examples, the light-darkcycle includes shorter periods of light and dark (for example periods oflight and dark from about 0.5 to about 4 hours). Such light-dark cyclesare referred to herein as “fluctuating” light cycles. In some examples,the fluctuating light cycles are not constant, for example the periodsof light are not all of the same length and/or the periods of dark arenot all of the same length. An exemplary fluctuating light-dark cycle isshown in FIG. 6A and an exemplary regular light-dark cycle is shown inFIG. 6B. In both the standard (or “regular”) light-dark cycles and thefluctuating light-dark cycles, the light intensity may be graduallyincreased and/or decreased during the light or dark phase, such that thelight-dark designation is not an all-or-none state. In some examples,the light-dark cycle is designed to provide a selected amount of light(μmoles of photons) over the total light-dark cycle. In some examples,the amount of light provided over a period of about 12-13 hours is about14,000-16,000 μmoles of photons. Photobioreactors which can beprogrammed for the desired light-dark cycle and light intensity arecommercially available (e.g., Phenometrics, Inc., Lansing, Mich.; PhotonSystem Instruments, Drasov, Czech Republic; Qubit Systems, Kingston,Ontario, Canada).

Production of increased biomass is useful for production of productsfrom plants or algae, such as lipids useful for biofuel. Methods ofextracting lipid from cells (such as plant or algae cells) are wellknown to one of ordinary skill in the art. In some examples, the cellsare lysed, for example by sonication or mechanical disruption (forexample using a French press or glass beads). Suitable methods for lipidextraction include, but are not limited to hexane solvent extraction,Soxhlet extraction, supercritical fluid extraction, extraction/expellerpress, and ultrasonic-assisted extraction. See, e.g., Brennan andOwende, Renewable and Sustainable Energy Reviews 14:557-577, 2010. Otherproducts that can be obtained from the disclosed transgenic cellsinclude pigments, nutritional supplements (such as omega-3 fatty acids),food sources, high value lipids, carotenoids, food stuffs, and otherproducts.

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit the disclosure to the particular features or embodimentsdescribed.

EXAMPLE 1 Generation of PDH and HST Transgenic Algae

This example describes the production of transgenic algae cell linesexpressing heterologous PDH or HST proteins.

A chloroplast transformation vector was used to express the pdh or hstgene in algae cells. The pBA155 vector, which restores photosynthesisupon successful homologous recombination of the psbA gene (coding forthe D1 protein of photosystem II), along with the gene of interest(Minagawa and Crofts, Photosynth. Res. 42:121-131, 1994). Acodon-optimized yeast PDH encoding nucleic acid (SEQ ID NO: 1) or an A.thaliana HST-encoding nucleic acid (SEQ ID NO: 3) was inserted in thepBA155 vector, which expresses the inserted gene under the control of astrong promoter, atpA. FIGS. 2A and B show schematic diagrams of the PDHand HST plasmids, respectively.

The psbA-deficient Chlamydomonas reinhardtii strain CC-4147 was grown tolog phase in TAP media and coated onto sterile TAP plates. The cellswere bombarded with plasmid DNA coated with gold particles. Highpressure helium gas carrying the coated gold particles serve asprojectiles that bombard the algae cells, delivering the DNA to thecells. Successful transformation results from homologous recombinationof the plasmid DNA to the chloroplast genomic DNA, restoring the psbAgene activity (and therefore restoration photosynthesis). Wildtype-complement cells (WT-c) are cells in which the deletion mutantlacking the chloroplast encoded psbA gene is complemented with thetransforming plasmid containing just the psbA gene.

Positive colonies (able to grow photosynthetically, on high salt (HS)media) were picked and moved to fresh HS-Amp 50 plates. The freshlygrowing colonies were then picked and suspended in 50 μL of 5%CHELEX®-100 resin (Bio-Rad Laboratories, Hercules, Calif.) followed byheat denaturation for 10 minutes at 98° C. Then 2 μL of the extract wasused as a template for the PCR reaction, using the KOD polymerase kit.The reaction mix for a single reaction was 25 μL 2× buffer, 10 μL dNTPmix, 1.5 μL each of forward and reverse primers, 2.0 μL template, 1.0 μLKOD polymerase, and 9.0 μL water. The same set of primers was used forPDH and HST, since they were expressed under the control of the samepromoter and terminator (Forward primer: 5′-CTAGGCAGTGGCGCGATGAC-3′ (SEQID NO: 5); Reverse primer: 5′-GGCCGCTCTAGCTAGAACTAGTGG-3′ (SEQ ID NO:6)). The psbA gene was also amplified as a control (Forward primer:5′-ATGACAGCAATTTTAGAACGTCG-3′ (SEQ ID NO: 7), Reverse primer:5′-TAGAACGTCGTGAAAATTCTAGCCTATGG-3′ (SEQ ID NO: 8)). PCR conditions were2 minutes at 94° C., 35 cycles of 10 seconds at 98° C., 30 seconds at56° C. and 2.5 minutes at 68° C., followed by 6 minutes at 68° C. PCRproducts were detected by ethidium bromide staining on 0.8-1% agarosegels. Multiple lines positive for PDH or HST were identified (FIG. 3A).

Expression of PDH was tested with RT-PCR. RNA was extracted from 5 mL ofcell culture pelleted by centrifuging for 5 minutes at room temperature.The pellet was frozen in liquid nitrogen and processed for RNA isolationor stored at −80° C. for later use. TRIZOL® reagent (1 mL,Invitrogen/Life Technologies, Carlsbad, Calif.) was added to the cellpellet, vortexed until homogenous and incubated at room temperature for5 minutes. Then 200 μL of chloroform was added, vortexed for 15 seconds,and incubated at room temperature for 3 minutes. The sample wascentrifuged for 15 minutes at 4° C. to eliminate debris and the aqueousphase was transferred to a fresh tube. The sample was then mixed with500 μL of isopropanol and incubated at room temperature for 10 minutes.The RNA was pelleted by centrifugation at 4° C. for 10 minutes, washedonce with 1 mL 80% ethanol, and repelleted by centrifuging for 5 minutesat 4° C. The RNA was air dried and resuspended in 100 μL water at 55° C.for 10-15 minutes.

The isolated RNA was treated with DNase for 1 hour at 37° C. followed byinactivation of the DNase. cDNA was synthesized with 10 μL of the RNAmix using gScript™ cDNA Super Mix (Quanta Biosciences, Gaithersburg,Md.). RT-PCR of PDH was carried out with forward primer5′-CCGCTGAAAAAAGAAATACATAA-3′ (SEQ ID NO: 9) and reverse primer5′-CTTTTTTCAGCGGCCGAACGG-3′ (SEQ ID NO: 10). The reaction mix was 2 μLTaq buffer, 1 μL dNTP mix, 0.5 μL each primer, 2.0 μL template, 0.5 μLBlue Taq polymerase, and 13.5 μL water. PCR conditions were 3 minutes at94° C., 30 cycles of 30 seconds at 94° C., 30 seconds at 58° C., and 2.5minutes at 72° C., followed by 5 minutes at 72° C. Samples (20 μL) wereloaded on 0.8-1% agarose gels and products detected by ethidium bromidestaining. The expected PDH amplification fragment was about 450 bp.Expression of PDH in five independent cell lines was confirmed by RT-PCR(FIG. 3B).

EXAMPLE 2 Characterization of PDH and HST Transgenic Algae

This Example describes characterization of non-photochemical quenching(NPQ), quinone reoxidation kinetics, and cell growth of transgenic algaeexpressing heterologous PDH or HST proteins.

Non-photochemical quenching (NPQ) was measured in wild type (WT) algae,four HST transgenic lines, and three PDH transgenic lines. For quinonereoxidation kinetics and fluorescence induction studies, cellsuspensions of the complemented WT and transgenic Chlamydomonas cellswere adjusted to a chlorophyll (Chl) concentration of about 5 μg Chl/mL.The protocol to measure the quinone reoxidation kinetics, measures F₀,before executing a single-turnover flash, and the fluorescence decay wasmonitored for 100 seconds. For fluorescence induction, the protocolmeasured F₀ followed by slow induction (˜750 μmoles at 650 nm). Thecells were dark adapted for 10 minutes prior to the experiment. Thequinone reoxidation kinetics were fit using the Origin softwareaccording to Vass et al. (Biochemistry 38: 12786-12794, 1999).

As shown in FIG. 4, the transgenic algae had reduced minimalfluorescence (F₀) and maximal fluorescence (F_(m)) as compared to the WTcomplement algae. This indicates that NPQ was reduced in the transgenicalgae and suggests the presence of an excess pool of oxidized PQ. Thekinetics of quinone reoxidation were also increased (FIGS. 5A-B) in PDHcell lines, further supporting the presence of an excess pool of PQ. Thelife-time component (T2) was faster in the PDH cell lines compared tothe WT line, while the T1 and T3 rate constants were similar between WTand transgenic cells (Table 2). The T2 component represents theoxidation of PQH₂ (membrane PQ pool) not bound to the Q0 site (theoxidation site at the Cytv6f complex) at the time of the actinic flash.A faster T2 component represents an overall increase in the kineticsachieved by overcoming the diffusional constraints with an increased PQpool size.

TABLE 2 Summary of chlorophyll fluorescence decay life-time componentsand their amplitudes Cell Line T1 (μs)/A1 (%) T2 (ms)/A2 (%) T3 (s)/A3(%) WT-complement 277/99.2 210/0.31 6/0.46 PDH1-4 262/99.2 100/0.336/0.33 PDH2-16 296/99.5  70/0.25 4.4/0.25 

To determine whether transgenic algae lines with elevated PQ levelsperformed as well as WT algae in fluctuating environments, the growth ofthe cell lines was measured in a Phenometric ePBR (Phenometric, Inc.,Lansing, Mich.) programmed for a fluctuating light cycle (FIG. 6A) or aregular light-dark cycle (FIG. 6B). The lowest and highest intensities(0 and 2000 μmole photons, respectively) were the same in thefluctuating profile and the regular profile. The fluctuating light cyclewas 13 hours (rather than 12 hours in the regular light-dark cycle) toprovide similar amounts of light during the day (14,625 μmole photonsfor fluctuating and 15,360 μmole photons for regular). The transgenicPDH cell lines had 3-5-fold higher biomass accumulation yield than theWT cells in the fluctuating light profile over a period of 12 days (FIG.7). Both the growth rate and the total biomass accumulation wereincreased in the PDH transgenic cell lines.

The results presented in this Example indicate that the PQ pool size wasincreased in PDH transgenic cell lines as compared to the WT cells. Thetransgenic cells also exhibited increased growth in fluctuating lightconditions compared to the WT cells.

EXAMPLE 3 Generation of PDH/HST Transgenic Algae

This example describes the production of transgenic algae cell linesexpressing heterologous PDH and HST proteins.

A chloroplast transformation vector was used to express both the pdh andhst genes in algae cells. The pBA155 vector, which restoresphotosynthesis upon successful homologous recombination of the psbA gene(coding for the D1 protein of photosystem II), along with the gene ofinterest (Minagawa and Crofts, Photosynth. Res. 42:121-131, 1994) wasutilized, as described in Example 1. Both a codon-optimized yeast PDHencoding nucleic acid (SEQ ID NO: 1) and an A. thaliana HST-encodingnucleic acid (SEQ ID NO: 3) were inserted in the pBA155 vector, each ofwhich were expressed under the control of a strong promoter, atpA (FIG.8).

The psbA-deficient Chlamydomonas reinhardtii strain CC-4147 wastransformed as described in Example 1. Positive colonies (able to growphotosynthetically on high salt (HS) media) were picked and moved tofresh HS-Amp 50 plates. Colonies were analyzed for presence of PDH andHST by PCR as described in Example 1. Multiple lines positive for bothPDH and HST were identified (FIG. 9). Two cell lines (PH-6 and PH-14)were selected for use in subsequent experiments.

EXAMPLE 4 Characterization of PDH/HST Transgenic Algae

This Example describes characterization of chlorophyll fluorescencekinetics of transgenic algae expressing both heterologous PDH and HSTproteins.

Fluorescence induction curves of algae samples normalized to 5 μg/μl Chlcontent were measured with and without 10 μM atrazine. Based on analysisof Chl fluorescence kinetics (FIGS. 10 and 11), a reduction in variableChl fluorescence (F_(v)=F_(m)−F_(o)) and the F_(v)/F_(m) (maximum) ratiowas observed in the PDH/HST transgenic lines both in the absence andpresence of atrazine (which binds to Qb site inhibiting photosystem IIelectron transfer) following induction with sub-saturating lightrelative to wild type. Without being bound by theory, these results areconsistent with both an increase in PQ levels and a disconnection of thelight harvesting antenna from the photosystem II reaction center,respectively. The latter observation is also consistent with an apparentreduction in light harvesting antenna size potentially increasing thequantum yield of photosynthesis (Yaakoubd et al., PhotosynthesisResearch 74:251-257, 2002; Kurreck et al., Photosynthesis Research63:171-182, 2000; Perrine et al., Algal Research 1:134-142, 2012).

In view of the many possible embodiments to which the principles of thedisclosure may be applied, it should be recognized that the illustratedembodiments are only examples and should not be taken as limiting thescope of the invention. Rather, the scope of the invention is defined bythe following claims. We therefore claim as our invention all that comeswithin the scope and spirit of these claims.

We claim:
 1. A transgenic photosynthetic cell comprising heterologousnucleic acids encoding: (i) a prephenate dehydrogenase (PDH) protein;and (ii) a homogentisate solanesyl transferase (HST) protein.
 2. Thetransgenic cell of claim 1, wherein the heterologous nucleic acidencoding the PDH protein comprises a nucleic acid sequence having atleast 90% identity to SEQ ID NO:
 1. 3. The transgenic cell of claim 1,wherein the heterologous nucleic acid encoding the PDH protein encodes aPDH protein comprising an amino acid sequence at least 90% identical toSEQ ID NO:
 2. 4. The transgenic cell of claim 1, wherein theheterologous nucleic acid encoding the HST protein comprises a nucleicacid sequence having at least 90% identity to SEQ ID NO:
 3. 5. Thetransgenic cell of claim 1, wherein the heterologous nucleic acidencoding the HST protein encodes an HST protein comprising an amino acidsequence at least 90% identical to SEQ ID NO:
 4. 6. The transgenic cellof claim 1, wherein the heterologous nucleic acid is expressed under thecontrol of a constitutive promoter.
 7. The transgenic cell of claim 1,wherein the heterologous nucleic acid is in an expression vector.
 8. Thetransgenic cell of claim 7, wherein the expression vector comprises aplasmid.
 9. The transgenic cell of claim 1, wherein the transgenic cellis a transgenic plant cell or a transgenic alga cell.
 10. The transgeniccell of claim 9, wherein the alga cell is a Chlamydomonas alga cell. 11.The transgenic cell of claim 1, wherein the transgenic cell hasincreased amounts of plastoquinone as compared to a control cell.
 12. Amethod of producing a photosynthetic cell comprising an increased amountof plastoquinone compared to a control cell, comprising expressing inthe cell heterologous nucleic acids encoding: (i) a prephenatedehydrogenase (PDH) protein; and (ii) a homogentisate solanesyltransferase (HST) protein.
 13. The method of claim 12, wherein theheterologous nucleic acid encoding the PDH protein comprises a nucleicacid at least 90% identical to SEQ ID NO:
 1. 14. The method of claim 13,wherein the heterologous nucleic acid encoding the PDH protein encodes aPDH protein comprising an amino acid sequence at least 90% identical toSEQ ID NO:
 2. 15. The method of claim 12, wherein the heterologousnucleic acid encoding the HST protein comprises a nucleic acid at least90% identical to SEQ ID NO:
 3. 16. The method of claim 15, wherein theheterologous nucleic acid encoding the HST protein encodes an HSTprotein comprising an amino acid sequence at least 90% identical to SEQID NO:
 4. 17. The method of claim 12, wherein the cell is a plant cellor an alga cell.
 18. The transgenic cell of claim 17, wherein the algacell is a Chlamydomonas alga cell.
 19. A method of increasing productionof biomass, comprising cultivating the transgenic cell of claim 1 underconditions sufficient to produce biomass, wherein the cultivatedtransgenic cell produces increased biomass as compared to a control. 20.The method of claim 19, wherein the conditions sufficient to producebiomass comprise a light-dark cycle.
 21. The method of claim 20, whereinthe light-dark cycle comprises: at least one period of light of about8-12 hours and at least one period of dark of about 12-16 hours; or atleast one period of light of about 30 minutes to 3 hours and at leastone period of dark of about 30 minutes to 3 hours.
 22. The method ofclaim 1, wherein the culture comprising the transgenic cell is grown ina bioreactor or a raceway.